[A bioluminescent solid phase for immunoassays and the detection of nucleic probes].
A luminescent adsorbent constituted of bacterial luciferase, FMN oxidoreductase and a protein, such as an antibody or an oligonucleotide coimmobilized on Sepharose, has been used to detect a label enzyme (Glucose 6 phosphate dehydrogenase). The label enzyme, bound to the solid phase, produces NADH and start an enzymatic chain reaction leading to light emission. The dehydrogenase, which is not bound to the solid phase, produces NADH in solution which is rapidly oxidized by a scavenger system (lactate dehydrogenase plus pyruvate) and thus does not participate in light emission. Using this solid phase, binding assays do not require separation of the excess of label, and the assay protocol is limited to the addition of sample, and luminescent reagents. The authors have used this solid phase for rapid immunoassays of haptens and proteins but also for the rapid quantitation of DNA sequences obtained by enzymatic amplification catalysed by a thermostable DNA polymerase.
Nicolas
J C
JC
INSERM Unité 58, Montpellier.
Térouanne
B
B
Balaguer
P
P
Boussioux
A M
AM
Crastes de Paulet
A
A
D038181:FMN Reductase; D005954:Glucosephosphate Dehydrogenase; D007124:Immunoenzyme Techniques; D008156:Luciferases; D008163:Luminescent Measurements; D015336:Molecular Probe Techniques; D009247:NADH, NADPH Oxidoreductases
D005954:Glucosephosphate Dehydrogenase; D008156:Luciferases; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases
Une phase solide bioluminescente pour les dosages immunologiques et la détection de sondes nucléiques.